Electrospun PNIPAAm/PCL Fiber Mats for Aligned Cell Sheets

ABSTRACT

The present invention provides compositions comprising aligned fibers of electrospun PNIPAAm and poly (ϵ-caprolactone) (PCL) (denoted PNIPAAm/PCL fibers). The PNIPAAm/PCL compositions enable enhanced growth and detachment of intact anisotropic cell sheets. The compositions do not require chemical modification or resource-intensive techniques, thus saving time and expense, and have the potential to generate tissue-specific, aligned cell sheets for transplant studies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/528,020, filed Jun. 30,2017, which is hereby incorporated byreference herein in its entirety.

BACKGROUND OF THE INVENTION

Cell alignment, which can influence developmental and physiologicalprocesses, is driven by biophysical cues, particularly matrixnanotopography (Kim et al., 2012, J. Cell Biol., 197:351-360). To bettermimic native cell microenvironments in vitro, techniques to generatesurface anisotropy, from electrospinning fibers (Lim et al., 2010,Biomaterials, 31:9031-9039; Levorson et al., 2013, Biomed. Mater.,8:014103) to micropatterning to photolithography, have been widelydeveloped and applied to anisotropic tissues, particularly muscle andnervous tissue. Both micropatterning and photolithography can be used todesign surfaces with high resolution but are resource-intensive,requiring microprinters and clean rooms (Falconnet et al., 2006,Biomaterials, 27:3044-3063). Electrospinning, on the other hand, is arelatively simple and inexpensive technique to fabricate polymer nano-and micro-fibers that can be seeded with cells (Doshi et al., 1993,Conference Record of the 1993 IEEE Industry Applications Society AnnualMeeting, 3:1698-1703; Pham et al., 2006, Tissue Eng., 12:1197-1211).Electrospun fibers are extracellular matrix-mimicking in that theyprovide a 3-dimensional fibrous microenvironment. Aligned electrospunfibers have been successful as scaffolds for generating nervous andbeating cardiac tissues that can be implanted into animal models (Kai etal., 2014, Acta Biomater., 10:2727-2738; Wang et al., 2012,Biomaterials, 33:9188-9197). These transplants, however, are ratherlimited in that they are only 1-2 cell layers thick, which can severelylimit function depending on tissue type, and the material component mayelicit a host response upon implantation (Anderson et al., 1996,Biomaterials Science: an Introduction to Materials in Medicine, Hostreactions to biomaterials and their evaluation, Academic Press).

Cell sheeting is a technique to generate biomaterial-free, tissue-likeconstructs for transplant. Teruo Okano pioneered the re-purposing ofthermosensitive poly(N-isopropylacrylamide) (PNIPAAm) as a surfacecoating to enable cell sheeting in vitro (Yamada et al., 1990, Makromol.Chem., Rapid Commun., 11:571-576). PNIPAAm undergoes a rapidcoil-to-globule transition at its lower critical solution temperature(LCST) of 32° C. that determines how the hydrophilic and hydrophobicdomains interact with water. Below 32° C., PNIPAAm readily dissolves inwater; above 32° C., PNIPAAm's hydrophilic domains are sequestered andPNIPAAm precipitates in aqueous solutions (Pelton, 2010, J. ColloidInterface Sci., 348:673-674). Thus, for cells grown on PNIPAAm-graftedtissue culture plates, cell sheet detachment is possible when theincubation temperature is lowered below the LCST: PNIPAAm expands,forcing the cell sheet to detach without perturbing cell-cell andcell-ECM adhesions. Using this technique, cells sheets have beengenerated for transplantation to the heart, cornea, and kidney (Yang etal., 2005, Biomaterials, 26:6415-6422, Yamato et al., 2007, Prog. Polym.Sci., 32:1123-1133). Yet generating aligned cell sheets has beenchallenging. Although grafting hydrophilic domains to PNIPAAm-graftedplates spatially controls cell attachment, leading to cell alignment,this approach required chemical synthesis and photolithographypatterning (Takahashi et al., 2011, Biomaterials, 32:8830-8838;Takahashi et al., 2011, Biomacromolecules, 12:1414-1418).

Thus, there is a need in the art for improved materials and methods forgenerating aligned cell sheets for various applications, includingtissue graft therapy. The present invention addresses this unmet need.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a fiber mat comprisingpoly(N-isopropylacrylamide) (PNIPAAm) and poly(caprolactone) (PCL),wherein the ratio of PNIPAAm to PCL is between 50% (1:1 PNIPAAm:PCL) to99% (99:1 PNIPAAm:PCL).

In one embodiment, the ratio of PNIPAAm to PCL is 90% (9:1 PNIPAAm:PCL).In one embodiment, the ratio of PNIPAAm to PCL is 75% (3:1 PNIPAAm:PCL).In one embodiment, the fiber mat has fibers with a diameter betweenabout 1 and 3 μm. In one embodiment, the fiber mat has fibers formedfrom a PNIPAAm core and a PCL shell. In one embodiment, the fiber mathas PNIPAAm fibers and PCL fibers. In one embodiment, the fiber mat hasfibers arranged substantially in parallel.

In another aspect, the present invention relates to a method of makingan anisotropic cell sheet, comprising the steps of: electrospinning asolution comprising poly(N-isopropylacrylamide) (PNIPAAm) andpoly(caprolactone) (PCL) to generate a fiber mat having fibers insubstantially parallel alignment; culturing cells on the fiber mat in anenvironment above about 32° C. to form an anisotropic sheet of cellsattached to the fiber mat; and introducing the fiber mat to an aqueousenvironment below about 32° C. to release an intact anisotropic sheet ofcells.

In one embodiment, the solution comprises a PNIPAAm and PCL mixturehaving a PNIPAAm to PCL ratio of between 50% (1:1 PNIPAAm:PCL) to 99%(99:1 PNIPAAm:PCL). In one embodiment, the PNIPAAm to PCL ratio is 90%(9:1 PNIPAAm:PCL). In one embodiment, the PNIPAAm to PCL ratio is 75%(3:1 PNIPAAm:PCL). In one embodiment, the solution comprises 10% to 20%wt/v of the PNIPAAm and PCL mixture dissolved in a mixture of methanoland chloroform.

In one embodiment, the electrospinning is performed using a rotatingmandrel. In one embodiment, the fibers have a PNIPAAm core and a PCLshell. In one embodiment, the fibers comprise PNIPAAm fibers and PCLfibers. In one embodiment, the fibers have a diameter between about 1and 3 μm.

In one embodiment, the fiber mat is wetted with an aqueous solutionabove about 32° C. prior to the step of culturing cells. In oneembodiment, the fiber mat is pretreated with a cell attachment enhancingcomposition prior to the step of culturing cells. In one embodiment, theattachment enhancing composition comprises at least one componentselected from the group consisting of: Matrigel, fetal bovine serum,gelatin, chitosan, fibronectin, collagen, poly-1-lysine, and laminin.

In one embodiment, the cells are selected from the group consisting of:urothelial cells, mesenchymal cells, muscle cells, myocytes,fibroblasts, chondrocytes, adipocytes, fibromyoblasts, ectodermal cells,hepotocytes, Islet cells, parenchymal cells, osteoblasts, nerve cells,and stem cells. In one embodiment, the fiber mat is introduced to anaqueous environment below about 32° C. by immersion or by rinsing.

In another aspect, the present invention relates to an anisotropic cellsheet formed by the method of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of embodiments of the invention willbe better understood when read in conjunction with the appendeddrawings. It should be understood that the invention is not limited tothe precise arrangements and instrumentalities of the embodiments shownin the drawings.

FIG. 1A through FIG. 1C depict results from example experimentsdemonstrating PNIPAAm/PCL fibers. (FIG. 1A) Scanning electron microscopy(SEM) images of electrospun PNIPAAm/PCL fibers. Percentage in upper leftindicates PNIPAAm content. Scalebars (black bars, bottom left) are 10μm. (FIG. 1B) Average fiber diameter determined using OrientationJ.(FIG. 1C) Fiber orientation index determined using OrientationJ. *p<0.05compared to 0% PNIPAAm fibers. FIG. 2A through FIG. 2C depict resultsfrom example experiments demonstrating chemical structures and FourierTransform Infrared spectroscopy (FTIR) results. Depicted are chemicalstructures of (FIG. 2A) PCL and (FIG. 2B) PNIPAAm. (FIG. 2C) FTIRspectra of PNIPAAm/PCL fibers. Dashed lines indicate absorption peaks.

FIG. 3A through FIG. 3C depict results from example experimentsdemonstrating PNIPAAm dissolution from PNIPAAm/PCL fibers. (FIG. 3A)PNIPAAm/PCL mass loss in water. PNIPAAm/PCL fiber (FIG. 3B) area percentchange and (FIG. 3C) axes (relative to fiber direction) length percentchange following PNIPAAm dissolution. # p<0.05 compared to all othergroups.

FIG. 4A and FIG. 4B depict results from example experimentsdemonstrating hydrophilicity of PNIPAAm/PCL fibers. (FIG. 4A) Advancingwater contact angle on PNIPAAm/PCL fibers. (FIG. 4B) Representativeimages of water droplet on fibers. White dashed lines indicate fiberedge. *p<0.05 compared to 0% PNIPAAm fibers. & p<0.05 compared to 100%PNIPAAm fibers.

FIG. 5A through FIG. 5C depict results from example experimentsdemonstrating cell viability and cell alignment on PNIPAAm/PCL fibers.(FIG. 5A) Cell viability relative to 0% PNIPAAm fibers determined by3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay. *p<0.05 compared to 0% PNIPAAm fibers. (FIG. 5B)Representative images of fibroblasts seeded on PNIPAAm/PCL fibers withactin (left) and actin/DAPI overlays (right). Scalebars are 200 (FIG.5C) Insets from 90% PNIPAAm images, as indicated by white dashed-box in(FIG. 5B). Scalebar is 50*p<0.05 compared to 0% PNIPAAm fibers.

FIG. 6A through FIG. 6E depict results from example experimentsdemonstrating cell sheet detachment. (FIG. 6A) Cell sheets detached from90% PNIPAAm fibers using room temperature medium; scalebar is 1 cm.(FIG. 6B, FIG. 6C) Cell sheet viability was confirmed with calcein, AMlive-staining. (FIG. 6D, FIG. 6E) Corresponding phase contrast images of(FIG. 6B, FIG. 6C); white arrows indicate residual PCL. Scalebars are(FIG. 6B, FIG. 6D) 400 μm and (FIG. 6C, FIG. 6E) 200 μm.

FIG. 7 depicts absorption peaks and assignments for PNIPAAm and PCL.

FIG. 8 depicts an illustration summarizing PNIPAAm/PCL fibers and cellsheeting, under various temperature conditions.

FIG. 9 depicts results from example experiments demonstrating thenormalized frequency plotted as a function of the cell orientationdegree. 3T3 fibroblasts were seeded on PNIPAAm/PCL fibers, grown for 24hours, and then stained with phalloidin to visualize actin. Images wereanalyzed in Image J to determine cell orientation. Cells were spread andclearly aligned on PNIPAAm/PCL fibers but not on 100% PNIPAAm fibers.

FIG. 10 depicts the construction of stand-alone PNIPAAm/PCL fiber bottomwells.

FIG. 11 depicts a series of images of PNIPAAm/PCL fibers (at variouspercentage of PNIPAAM content) before and after wetting.

FIG. 12 depicts the results of experiments depicting the advancing watercontact angle of dry and wetted PNIPAAm/PCL fibers at 0%, 90% and 100%PNIPAAm content.

FIG. 13 depicts the results of experiments differentiating mouseembryonic stem cells (mESCs) into cardiomyocytes on PNIPAAm/PCL fibersheets. The top row depicts four PNIPAAm/PCL fiber sheets having thesame ratio of PNIPAAm:PCL (90:1). The number in the top left cornerindicates the alignment scale from 0 (unaligned) to 1 (perfect parallelalignment). The bottom row depicts mESCs differentiated on the mats,with cTnT marker being used to identify differentiation into cardiaccells. Cell orientation is observed in cultures on mats having alignmentscales above 0.50.

FIG. 14 depicts the results of experiments measuring the synchronizationof mESCs differentiated into beating cardiomyocytes on PNIPAAm/PCL fibersheets shown in FIG. 13. The top row shows an 8 day culture of mESCs ona PNIPAAm/PCL fiber sheet having an alignment scale of 0.23. The bottomrow shows an 8 day culture of mESCs on a PNIPAAm/PCL fiber sheet havingan alignment scale of 0.86. Beating is measured by fluorescent calciumreporter dye. The results demonstrate the differentiation on the highlyaligned fiber sheet has synchronized beating.

FIG. 15 depicts additional data processing from the results of FIG. 14.The bottom left images are heatmap signals evaluating the beating of theentire culture on the fiber sheet. The top heatmap shows signalsoriginating at different time points in the culture on the fiber sheethaving an alignment scale of 0.23. The bottom heatmap shows signalsoriginating at substantially the same time in the culture on the fibersheet having an alignment scale of 0.86. The bottom right image is a bargraph generated from the quantification of the signal timings in theheatmaps (y-axis label: time for point arrival-median absolutedeviation).

FIG. 16A depicts a time series image analyzed in PIVlab showing motionvectors for the contraction directionality of mCherry expressing mESCsdifferentiated into cardiomyocytes on aligned PCL fibers. FIG. 16B is achart quantifying the relative frequency of each cardiomyocytecontraction direction, demonstrating that on aligned fibers, cardiaccontraction has a preferred contraction direction, which is the fiberdirection.

FIG. 17 depicts a schematic and actual testing setup of cantileverbending. Cardiomyocyte contraction causes the cantilever to moveupwards, and the resulting change in length is recorded. Scale bar=10mm. Cantilever bending was only detected in cardiomyocytes that weredifferentiated on aligned fiber scaffolds and were beatingsynchronously.

DETAILED DESCRIPTION

The present invention provides compositions comprising aligned fibers ofelectrospun PNIPAAm and poly (ϵ-caprolactone) (PCL). The PNIPAAm/PCLcompositions enable enhanced growth and detachment of intact anisotropiccell sheets. The compositions demonstrate an unexpected and significantbenefit in that they do not require chemical modification orresource-intensive techniques, thus saving time and expense, and havethe potential to generate tissue-specific, aligned cell sheets for awide variety of applications, including but not limited to tissue repairand drug screening.

Definitions

It is to be understood that the figures and descriptions of the presentinvention have been simplified to illustrate elements that are relevantfor a clear understanding of the present invention, while eliminating,for the purpose of clarity, many other elements typically found in theart. Those of ordinary skill in the art may recognize that otherelements and/or steps are desirable and/or required in implementing thepresent invention. However, because such elements and steps are wellknown in the art, and because they do not facilitate a betterunderstanding of the present invention, a discussion of such elementsand steps is not provided herein. The disclosure herein is directed toall such variations and modifications to such elements and methods knownto those skilled in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, exemplary methods andmaterials are described

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value,as such variations are appropriate to perform the disclosed methods.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

PNIPAAm/PCL Fiber Mats

The present invention relates in part to the unexpected discovery that

PNIPAAm and PCL in solution can be electrospun into mats of uniquelystructured fibers in substantially parallel alignment. Further, it isdescribed herein that mats formed from electrospun PNIPAAm/PCL atcertain ratios are capable of supporting cell cultures forming intactcell sheets. The electrospun mats can include PNIPAAm fibers alongsidePCL fibers, and can include fibers having a PNIPAAm core and a PCLsheath.

Electrospinning is a process exploiting the interactions between anelectrostatic field and a conducting fluid. When an externalelectrostatic field is applied to a conducting fluid (e.g., asemi-dilute polymer solution or a polymer melt), a suspended conicaldroplet is formed, whereby the surface tension of the droplet is inequilibrium with the electric field. As it reaches a grounded target,the material can be collected as an interconnected web containingrelatively fine, i.e. small diameter, fibers. The resulting films (ormembranes) from these small diameter fibers have very large surface areato volume ratios and small pore sizes.

Electrospinning involves the spinning of non-woven fabric of solutionsor melts using very high voltages. The solvent is pumped to a needle,called the spinneret, where a very high voltage is applied. If thisvoltage is high enough, the repelling charges will be stronger than thesurface tension will keep the solution together, and generate a chargedjet from the Taylor cone at the needle tip. By placing a differentlycharged target at a defined distance, these cones will start to depositvery thin fibers onto the target.

The present invention combines the temperature-dependent dissolutionaspects of PNIPAAm with cell attachment aspects of PCL to electrospinaligned fiber mats capable of supporting the growth and intact releaseof anisotropic cell sheets. Accordingly, the present invention providesPNIPAAm and PCL blends for electrospinning. In various embodiments, theblends comprise a PNIPAAm to PCL ratio of between 50% (1:1 PNIPAAm:PCL)and 99% (99:1 PNIPAAm:PCL). In some embodiments, the blends comprise aPNIPAAm to PCL ratio of between 75% (3:1 PNIPAAm:PCL) and 90% (9:1PNIPAAm:PCL). In various embodiments, the blends comprise a PNIPAAm toPCL ratio of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, or 90%. The PNIPAAm and PCL can be dissolved in anysuitable solvent. Typical solvents include N,N-Dimethyl formamide (DMF),tetrahydrofuran (THF), methylene chloride, methanol, dioxane, ethanol,hexafluoroisopropanol (HFIP), chloroform, glacial acetic acid, water,and combinations thereof. For example, in one embodiment the solvent isa mixture of methanol and chloroform. In one embodiment, the PNIPAAm/PCLblend comprises between 10 and 20% wt/v of a PNIPAAm/PCL compositiondissolved in a mixture of methanol and chloroform.

In certain embodiments the mixture of methanol and chloroform having aratio of about 10:1 to about 1:10 of methanol to chloroform. In oneembodiment, the PNIPAAm/PCL is dissolved in a 1:3 mixture of methanoland chloroform.

The PNIPAAm/PCL blends are amenable to modification to enhance fibergeneration, fiber structure, cell attachment, cell growth, and the like.For example, in one embodiment, the blends can optionally contain a saltto create an excess charge effect to facilitate the electrospinningprocess. Examples of suitable salts include NaCl, KH₂PO₄, K₂HPO₄, KIO₃,KCl, MgSO₄, MgCl₂, NaHCO₃, CaCl₂ or mixtures of these salts.

In one embodiment, the blends can include biopolymers, such asextracellular matrix proteins, while maintaining the PNIPAAm to PCLratio. Exemplary biopolymers include but are not limited to collagen,fibrin, elastin, gelatin, fibrinogen, thrombin, laminin, chondroitinsulfates, heparins, hyaluronic acid, alginate, dextran, pectin, andchitosan. In certain embodiments, the biological component comprisesamino acids, peptides, proteins, carbohydrates, lipids, nucleic acids,glycoproteins, minerals, lipoproteins, glycolipids, glycoaminoglycans,and proteoglycans. In various embodiments, the blends can furthercomprise one or more synthetic material while maintaining the PNIPAAm toPCL ratio. The synthetic materials are preferably biologicallycompatible to support cell growth. Such polymers include but are notlimited to the following: poly(urethanes), poly(siloxanes) or silicones,poly(ethylene), poly(vinyl pyrrolidone), poly(-hydroxy ethylmethacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate),poly(vinyl alcohol), poly(acrylic acid), poly(ethylene-co-vinylacetate), poly(ethylene glycol), poly(methacrylic acid), polylactic acid(PLA), polyglycolic acids (PGA), poly(lactide-co-glycolides) (PLGA),nylons, polyamides, polyanhydrides, poly(ethylene-co-vinyl alcohol)(EVOH), poly(vinyl acetate) (PVA), polyvinylhydroxide, poly(ethyleneoxide) (PEO) and polyorthoesters or any other similar synthetic polymersthat may be developed that are biologically compatible. Polymers withcationic moieties can also be used, such as poly(allyl amine),poly(ethylene imine), poly(lysine), and poly(arginine). The polymers mayhave any molecular structure including, but not limited to, linear,branched, graft, block, star, comb, and dendrimer structures.

Method of Generating PNIPAAm/PCL Fibers Mats

As described elsewhere herein, the PNIPAAm/PCL blends are electrospun togenerate PNIPAAm/PCL fiber mats. The blends are electrospun onto arotating mandrel to generate mats of blended fibers alignedsubstantially in parallel. The conditions under which the blends arespun can be performed within any suitable range, such as those disclosedherein. For example, the electric field used in the electrospinningprocess can be in the range of about 1 to about 50 kV, more preferablyfrom about 5 to about 15 kV. The feed rate of the blend to the spinneretcan be in the range of about 1 to about 2 mL/hour. The working distanceof the spinneret to the rotating substrate can be in the range of about5 to about 15 cm, or more preferably about 10 to about 11 cm.

Persons skilled in the art will understand that the rotating substratetypically involves a mandrel mechanically attached to a motor, oftenthrough a drill chuck. In various embodiments, the motor rotates themandrel at a speed of between about 1 revolution per minute (rpm) toabout 40,000 rpm. In one exemplary embodiment, the rotation speed isbetween about 2500 rpm to about 3500 rpm.

The resultant PNIPAAm/PCL fiber mats comprise aligned fibers of between1 and 3 μm in diameter. As described elsewhere herein, the PNIPAAm coreenables temperature-dependent dissolution for controlled release ofcultured cells. In some embodiments, fibers comprising a PNIPAAm coreare protected by a PCL shell while enhancing cell attachment to thefibers. In other embodiments, PNIPAAm fibers are protected by adjacentPCL fibers. Post-electrospinning, the PNIPAAm/PCL fiber mats can betreated in any suitable manner, such as being cut into any variousshapes and sizes, sterilized, and stored for later use.

The PNIPAAm/PCL fiber mats are amenable to modification to enhance cellattachment, cell growth, and the like. In various embodiments, thePNIPAAm/PCL fiber mats can be modified with one or more functionalgroups for covalently attaching a variety of proteins (e.g., collagen)or compounds such as therapeutic agents. Therapeutic agents which may belinked to the fiber mats include, but are not limited to, analgesics,anesthetics, antifungals, antibiotics, anti-inflammatories,anthelmintics, antidotes, antiemetics, antihistamines, anti-cancerdrugs, antihypertensives, antimalarials, antimicrobials, antipsychotics,antipyretics, antiseptics, antiarthritics, antituberculotics,antitussives, antivirals, cardioactive drugs, cathartics,chemotherapeutic agents, a colored or fluorescent imaging agent,corticoids (such as steroids), antidepressants, depressants, diagnosticaids, diuretics, enzymes, expectorants, hormones, hypnotics, minerals,nutritional supplements, parasympathomimetics, potassium supplements,radiation sensitizers, a radioisotope, fluorescent nanoparticles such asnanodiamonds, sedatives, sulfonamides, stimulants, sympathomimetics,tranquilizers, urinary anti-infectives, vasoconstrictors, vasodilators,vitamins, xanthine derivatives, and the like. The therapeutic agent mayalso be other small organic molecules, naturally isolated entities ortheir analogs, organometallic agents, chelated metals or metal salts,peptide-based drugs, or peptidic or non-peptidic receptor targeting orbinding agents.

In various embodiments, the PNIPAAm/PCL fiber mats can further bemodified to comprise one or more polysaccharides, includingglycosaminoglycans (GAGs) or glucosaminoglycans, with suitableviscosity, molecular mass, and other desirable properties. The term“glycosaminoglycan” is intended to encompass any glycan (i.e.,polysaccharide) comprising an unbranched polysaccharide chain with arepeating disaccharide unit, one of which is always an amino sugar.These compounds as a class carry a high negative charge, are stronglyhydrophilic, and are commonly called mucopolysaccharides. This group ofpolysaccharides includes heparin, heparan sulfate, chondroitin sulfate,dermatan sulfate, keratan sulfate, and hyaluronic acid. These GAGs arepredominantly found on cell surfaces and in the extracellular matrix.The term “glucosaminoglycan” is also intended to encompass any glycan(i.e. polysaccharide) containing predominantly monosaccharidederivatives in which an alcoholic hydroxyl group has been replaced by anamino group or other functional group such as sulfate or phosphate. Anexample of a glucosaminoglycan is poly-N-acetyl glucosaminoglycan,commonly referred to as chitosan. Exemplary polysaccharides that may beuseful in the present invention include dextran, heparan, heparin,hyaluronic acid, alginate, agarose, carageenan, amylopectin, amylose,glycogen, starch, cellulose, chitin, chitosan and various sulfatedpolysaccharides such as heparan sulfate, chondroitin sulfate, dextransulfate, dermatan sulfate, or keratan sulfate.

In one embodiment, the PNIPAAm/PCL fiber mats can further be modified tocomprise one or more natural or synthetic drug, such as nonsteroidalanti-inflammatory drugs (NSAIDs). In one embodiment, the fiber mats canfurther comprise antibiotics, such as penicillin. In one embodiment, thefiber mats can further comprise natural peptides, such asglycyl-arginyl-glycyl-aspartyl-serine (GRGDS), arginylglycylasparticacid (RGD), and amelogenin. In one embodiment, the fiber mats canfurther comprise proteins, such as chitosan and silk. In one embodiment,the fiber mats can further comprise sucrose, fructose, cellulose, ormannitol. In one embodiment, the fiber mats can further compriseextracellular matrix proteins, such as fibronectin, vitronectin,laminin, collagens, and vixapatin (VP12). In one embodiment, the fibermats can further comprise disintegrins, such as VLO4. In one embodiment,the fiber mats can further comprise decellularized or demineralizedtissue. In one embodiment, the fiber mats can further comprise syntheticpeptides, such as emdogain. In one embodiment, the fiber mats canfurther comprise nutrients, such as bovine serum albumin. In oneembodiment, the fiber mats can further comprise vitamins, such asvitamin B2, vitamin Ad, Vitamin D, Vitamin E, and Vitamin K. In oneembodiment, the fiber mats can further comprise nucleic acids, such asmRNA and DNA. In one embodiment, the fiber mats can further comprisenatural or synthetic steroids and hormones, such as dexamethasone,hydrocortisone, estrogens, and its derivatives. In one embodiment, thefiber mats can further comprise growth factors, such as fibroblastgrowth factor (FGF), transforming growth factor beta (TGF-β), andepidermal growth factor (EGF). In one embodiment, the fiber mats canfurther comprise a delivery vehicle, such as nanoparticles,microparticles, liposomes, viral and non-viral transfection systems.

Method of Generating Anisotropic Cell Sheets

The PNIPAAm/PCL aligned fiber mats of the present invention enableenhanced anisotropic cell sheet growth and detachment. The fiber matsserve as the substrate for cell attachment and proliferation, and uponincubation or rinsing of the fiber mats with aqueous solution below thecritical temperature of 32° C., the PNIPAAm portion of the fibersdissolute, the remaining PCL portion breaks down, and an intact cellsheet is released free of any fibers. In certain embodiments, thePNIPAAm/PCL fiber mats are wetted prior to seeding cells to improvefiber mat handling. Wetting can be accomplished using any suitableliquid media at a temperature above 32° C., such as water, phosphatebuffered saline, fetal bovine serum, and any typical cell culture media.

The PNIPAAm/PCL fiber mats are amenable to any suitable cell culture.Typical cell include, but are not limited to: urothelial cells,mesenchymal cells, especially smooth or skeletal muscle cells, myocytes(muscle stem cells), fibroblasts, chondrocytes, adipocytes,fibromyoblasts, and ectodermal cells, including ductile and skin cells,hepotocytes, Islet cells, cells present in the intestine, and otherparenchymal cells, osteoblasts and other cells forming bone orcartilage, nerve cells, and stem cells. Stem cells include but are notlimited to: embryonic stem cells, fetal stem cells, adult stem cells,induced pluripotent stem cells, mesenchymal stem cells, hematopoieticstem cells, neural stem cells, and epithelial stem cells. Selection ofcell types, and seeding of cells onto the fiber mats, will be routine toone of ordinary skill in the art in light of the teachings herein.

The anisotropic cell sheets are able to mimic characteristics of thecell origin tissues. For example, epithelial cells cultured on the fibermats can form epithelial tissue, liver cells cultured on the fiber matscan form liver tissue, kidney cells cultured on the fiber mats can formkidney tissue, endothelial cells cultured on the fiber mats can formendothelial tissue, skeletal muscle cells cultured on the fiber mats canform skeletal muscle tissue, cardiac muscle cells cultured on the fibermats can form cardiac muscle tissue, interstitial valvular cellscultured on the fiber mats can form valvular tissue, and the like.

The anisotropic cell sheets can be useful for wound care and tissueregeneration. In one aspect, the anisotropic cell sheets can be used asorgan or tissue grafts. The anisotropic cell sheets can be used to treatwounds or tissue damage resulting from trauma, disease, burns, ulcers,abrasions, lacerations, surgery, or other damage. The anisotropic cellsheets can also be used to treat internal soft tissue wounds or defectssuch as wounds in the amniotic sac, ulcers in the gastrointestinal tractor mucous membranes, gingival damage or recession, damaged or diseasedcardiac tissue, damaged or diseased skeletal muscle tissue, internalsurgical incisions or biopsies, and the like. Surgeons can use thesegrafts to cover and protect the area in need of treatment, totemporarily replace lost or damaged tissue, and to guide new tissuegeneration and healing into the damaged area. The anisotropic cellsheets may be secured to the treatment area using sutures, adhesives, oroverlaying bandages. The anisotropic cell sheets may be cut to match thesize of the treatment area, or may overlap the edges of the treatmentarea.

In some embodiments, grafted anisotropic cell sheets are also useful fordelivery of biologics, enzymes that activate drugs, protease inhibitors,and the like. The anisotropic cell sheets can include native cells aswell as nonnative cells that have the ability to express angiogenicgrowth factors and cytokines, secrete wound healing related cytokines,secrete collagen, and promote wound healing in vivo. The anisotropiccell sheets may also be embedded or conjugated with various factorswhich may be released at a graft site. These factors may include, butare not limited to epidermal growth factor (EGF), platelet derivedgrowth factor (PDGF), basic fibroblast growth factor (bFGF),transforming growth factor-β (TGF-β), and tissue inhibitors ofmetalloproteinases (TIMP), which have been shown to be beneficial inwound healing. Additional healing factors such as antibiotics,bacteriocides, fungicides, silver-containing agents, analgesics, andnitric oxide releasing compounds can also be incorporated into theanisotropic cell sheets.

In some embodiments, the anisotropic cell sheets can be used in cell- ortissue-based screening. In some embodiments, the screening can be forone or more infectious diseases such as viral infection or parasiticinfection. In some embodiments, the screening can be for injuries andsecondary injuries such as scarring and inflammation. In someembodiments, the screening can be for abnormalities such as atrophy andhypertrophy. In some embodiments, the screening can be for one or moremetabolic deficiencies. In some embodiments, the screening can be forone or more protein deficiencies. In some embodiments, the screening canbe for cancer, including the study of cancer initiation, progression, ormetastasis. In some embodiments, the anisotropic cell sheets can be usedin the study of the interaction of other cell types, such as cancercells, pathogen-bearing cells, pathogenic cells, immune cells,blood-derived cells, or stem/progenitor cells.

In some embodiments, the anisotropic cell sheets can be used for drugscreening or drug discovery. In some embodiments, an array, microarray,or chip can incorporate the anisotropic cell sheets for drug screeningor drug discovery. In some embodiments, an anisotropic cell sheet can besectioned and distributed to each well of a biocompatible multi-wellcontainer, wherein the container is compatible with one or moreautomated drug screening procedures and/or devices. The drug screeningor drug discovery can be used to research or develop drugs potentiallyuseful in any therapeutic area, including infectious diseases,hematology, oncology, pediatrics, cardiology, central nervous systemdisease, neurology, gastroenterology, hepatology, urology, infertility,ophthalmology, nephrology, orthopedics, pain control, psychiatry,pulmonology, vaccines, wound healing, physiology, pharmacology,dermatology, gene therapy, toxicology, and immunology.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the present invention andpractice the claimed methods. The following working examples thereforeare not to be construed as limiting in any way the remainder of thedisclosure.

Example 1 Electrospun Poly(N-Isopropyl Acrylamide)/Poly(Caprolactone)Fibers for the Generation of Anisotropic Cell Sheets

Cell alignment in muscle, nervous tissue, and cartilage is requisite forproper tissue function; however, cell sheeting techniques using thethermosensitive polymer poly(N-isopropyl acrylamide) (PNIPAAm) can onlyproduce anisotropic cell sheets with delicate and resource-intensivemodifications. Without wishing to be bound by any particular theory, itwas hypothesized that electrospinning, a relatively simple andinexpensive technique to generate aligned polymer fibers, could be usedto fabricate anisotropic PNIPAAm and poly(caprolactone) (PCL) blendedsurfaces that both support cell viability and permit cell sheetdetachment via PNIPAAm dissolution. Aligned electrospun PNIPAAm/PCLfibers (0%, 25%, 50%, 75%, 90%, and 100% PNIPAAm) were electrospun andcharacterized. Fibers ranged in diameter from 1-3 μm, and all fibers hadan orientation index greater than 0.65 (orientation index lower limit of0 for random orientation; upper limit of 1 for perfect alignment).Fourier transform infrared spectroscopy was used to confirm the relativecontent of PNIPAAm and PCL. For advancing water contact angle and massloss studies, only high PNIPAAm-content fibers (75% and greater)exhibited temperature-dependent properties like 100% PNIPAAm fibers,whereas 25% and 50% PNIPAAm fibers behaved similarly to PCL-only fibers.3T3 fibroblasts seeded on all PNIPAAm/PCL fibers had high cell viabilityand spreading except for the 100% PNIPAAm fibers. Cell sheet detachmentby incubation with cold medium was successful for 90% PNIPAAm fibers,which had a sufficient amount of PCL to allow cell attachment andspreading but not enough to prevent detachment upon PNIPAAm dissolution.This study demonstrates the feasibility of using anisotropic electrospunPNIPAAm/PCL fibers to generate aligned cell sheets that can potentiallybetter recapitulate anisotropic architecture to achieve proper tissuefunction.

The materials and methods employed in these experiments are nowdescribed.

Fabrication PNIPAAm/PCL Fibers

For PNIPAAm-only fibers, PNIPAAm (300 000 Da, Scientific PolymerProducts, Ontario, N.Y.) was dissolved 20% (wt/v) in methanol (FisherChemical, Pittsburgh, Pa.), as previously described (Lee et al., 2016,Adv. Healthcare Mater., 5:781-785). For PCL-only fibers, PCL (80 000 Da,Sigma-Aldrich, St Louis, Mo.) was dissolved 10% (wt/v) inhexafluoroisopropanol (Sigma-Aldrich). For PNIPAAm/PCL fibers, PNIPAAmand PCL (at ratios of 9:1, 3:1, 1:1, and 1:3, respectively) weredissolved 12-18% (wt/v) in a 1:3 mixture of methanol and chloroform(Sigma-Aldrich). All polymer solutions were dissolved by continuousstirring until clear and homogenous. To electrospin, a syringe pump (NewEra Pump Systems, Inc.) was used to dispense the polymer solutions froma 10 mL syringe with a 25 G blunted stainless steel needle at 2.0 mL h⁻¹for the PCL-only solution and 1.0 mL h⁻¹ for all PNIPAAm-containingsolutions. A high voltage supply (Gamma High Voltage, Ormond Beach,Fla.) was used to apply a charge of 5-15 kV (optimal charge determinedfor each solution, Table 1) to the needle to initiate jet formation.Fibers were deposited on a rotating grounded 7.6 cm diameter aluminumcollector. To obtain aligned fibers, the collector was rotated at2500-3200 rotations per minute (RPM, approximately 10.0-12.8 m s⁻¹). Theworking distance from the needle and to collector was set at 11 cm.PNIPAAm/PCL fibers are referred to by the percent PNIPAAm content (i.e.,75% PNIPAAm fibers comprise 75% PNIPAAm and 25% PCL). Similarly, “highPCL-content” refers to 0%, 25%, and 50% PNIPAAm and “highPNIPAAm-content” refers to 75%, 90%, and 100% PNIPAAm.

TABLE 1 PNIPAAm Total Polymer content (wt/vol) Solvent Charge (kV)  0%10% HFP 4.5-6.5  25% 12% 1:3 methanol:chloroform  7.0-10.5  50% 12% 1:3methanol:chloroform 8.5-9.5  75% 15% 1:3 methanol:chloroform  9.0-10.0 90% 18% 1:3 methanol:chloroform 13.0-15.0 100% 20% methanol 11.0-11.5

Characterization of PNIPAAm/PCL Fibers

Fiber Orientation and Diameter

PNIPAAm/PCL fibers were sputter coated with 12 nm platinum/palladium andimaged using a Zeiss Supra 40VP scanning electron microscope (SEM, 5kV). SEM images (n=9) were analyzed using NIH ImageJ software,specifically the OrientationJ (Rezakhaniha et al., 2012, Biomech. Model.Mechanobiol., 11:461-473; Püspöki et al., 2016, Adv. Anat., Embryol.Cell Biol., 219:69-93) and Diameter) (Hotaling et al., 2015,Biomaterials, 61:327-338) plug-ins to determine fiber orientation anddiameter, respectively. The fiber orientation index, S, was calculatedfrom angle distribution histograms using the following equation:(Ferdman et al., 1993, J. Invest. Dermatol., 100:710-716)

S=2<cos²(α)>−1

where α is the difference between an individual fiber angle and the meanangle of all fibers. S varies from 0 to 1, for perfectly random andperfectly aligned fibers, respectively.

Fourier Transform Infrared Spectroscopy

Fourier-transform infrared (FTIR) attenuated total reflectance (ATR) wasused to verify fiber polymer composition. Spectra of dry PNIPAAm/PCLfibers was collected over a range of wavelengths (400 cm⁻¹ to 3000 cm⁻¹)at a resolution of 2 cm⁻¹ using a Thermo Scientific Nicolet iS10 FT-IRspectrometer (Waltham, Mass.). Background spectra was collected prior toeach individual sample.

PNIPAAm Mass Loss

PNIPAAm/PCL fibers were cut (approximately 1 cm×1 cm, n=3) and weighedbefore being immersed in ultrapure water at room temperature. To ensurecomplete PNIPAAm dissolution, fibers were rinsed 3 times in 2 mL ofwater over 24 hours. Fibers were then dried under vacuum for 48 hoursbefore measuring their final weight. Percent mass lost was determined bysubtracting the final weight from the original weight. Fibers wereimaged before and after rinsing, and original and post-dissolution areaswere calculated in ImageJ. Percent contraction was determined bydividing the final area by the original area.

Advancing Contact Angle Measurement

Advancing contact angles were measured using a FTA-200 goniometer (FirstTen Angstroms, Portsmouth, Va.) to determine the relative hydrophobicityof dry (non-wetted) and wetted PNIPAAm/PCL fibers. Fibers were cut intosquares (approximately 1.75 cm×1.75 cm, n=3). Wetted PNIPAAm/PCL fibersquares were secured in CellCrown inserts and rinsed in water warmed to37° C. for 24 hours. Fibers were then dried in a vacuum oven at 35-55°,above the LCST of PNIPAAm and below the melting temperature of PCL.PNIPAAm/PCL fibers were placed on a heating platform to maintain thetemperature between 32-60° C., as measured by an infrared thermometerfor advancing water content angle analysis. Briefly, a drop of purifiedwater was deposited at 0.8 μL per second from a 10 mL syringe on thePNIPAAm/PCL fibers, and high resolution images were subsequentlycaptured. When possible, the contact angle was determined in the sessiledrop session mode in the instrument-associated software. Otherwise,advancing contact angle was calculated by manually defining the locationof the fiber plane and the drop's curvature.

Cell Studies

All cell studies were performed using NIH 3T3 fibroblasts purchased fromAmerican Type Culture Collection (Manassas, Va.), passages 15-25. 3T3fibroblasts were maintained in 10% fetal bovine serum (ThermoFisher) and1% penicillin/streptomycin (ThermoFisher) in low glucose Dulbecco'sModified Eagle's Medium with L-glutamine (Sigma-Aldrich). 3T3fibroblasts were passaged using trypsin/EDTA (Sigma-Aldrich).

PNIPAAm/PCL Fiber Sterilization and Protein Coating

PNIPAAm/PCL fibers and parafilm were cut into squares (1.75 cm×1.75 cmor 2.5 cm×2.5 cm) and sterilized by ultraviolet light, 30 minutes eachside. Fibers were secured using 12-well or 24-well CellCrown inserts(Scaffdex, Tampere, Finland) and parafilm (Bemis, Oshkosh,Wis.)—effectively making stand-alone PNIPAAm/PCL fiber bottom wells(FIG. 10)—to improve handling and prevent PNIPAAm fiber contraction uponwetting. Prior to coating, fibers were wetted with Dulbecco's PhosphateBuffered Saline (DPBS). Fibers were then coated with a 1:50 dilution ofGrowth-factor Reduced (GFR)-Matrigel (Corning, Corning, N.Y.) inDulbecco's Modified Eagle's Medium (DMEM). Fibers were also pre-treatedwith fetal bovine serum (FBS, ThermoFisher) for at least 10 minutesimmediately prior to cell seeding, as recommended by Haraguchi et al.(Haraguchi et al., 2012, Nat. Protoc., 7:850-858). All solutions werewarmed to 37° C. and fibers were kept on a hot plate in the tissueculture hood to ensure that PNIPAAm remained above its LCST.

Cell Viability on PNIPAAm/PCL Fibers 3T3 fibroblasts were seeded ontoPNIPAAm/PCL fibers (n=3) at 120,000 cells per cm². This cell density wasselected to ensure that the absorbance for the colorimetric assay was inthe linear range. 24 hours post-seeding, cells were rinsed and treatedwith the tetrazolium dye3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS, CellTiter 96® AQueous One Solution Cell Proliferation Assay,Promega, Madison, Wis.). Solution absorbance was measured at 490 nm on aBiotek Cytation 3 Cell Imaging Multi-Mode Reader. Each condition wasnormalized by solution absorbance of 3T3 fibroblast-seeded PCL-onlycontrols to determine relative cell attachment.

Cell Alignment on PNIPAAm/PCL Fibers

3T3 fibroblasts were seeded onto PNIPAAm/PCL fibers at 240,000 cells percm². 24 hours post-seeding, cells on PNIPAAm/PCL fibers were rinsed andfixed in 4% (v/v) paraformaldehyde for 15 minutes. Cells werepermeabilized with 0.2% (v/v) Triton X-100 for 10 minutes and thenstained with 6.6 μM rhodamine phalloidin (ThermoFisher). Following actinstaining, cell nuclei were stained with 300 nM4′,6-diamidino-2-phenylindole (DAPI, ThermoFisher). All solutions werewarmed to and fibers were incubated at 37° C., above PNIPAAm's LCST, asdescribed previously (Takahashi et al., 2011, Biomaterials,32:8830-8838). Cell-seeded fibers were dissembled from CellCrowninserts, placed on slides, and imaged on a Biotek Cytation 3 CellImaging Multi-Mode Reader incubated to 37° C. Actin images were analyzedusing NIH ImageJ software and OrientationJ plug-in to determine cellalignment.

Cell Sheet Detachment from PNIPAAm/PCL Fibers

3T3 fibroblasts were seeded onto PNIPAAm/PCL fibers at 630,000 cells percm² and allowed to grow for 4 days. Medium was changed every day. Priorto detachment, cells were stained with 5 μM calcein, AM (ThermoFisher)and nuclear stain Hoescht 33342 (ThermoFisher) for 30 minutes.Detachment was initiated by rinsing 5 times over 10-15 minutes with cold(approximately 4° C.) medium to dissolve and remove PNIPAAm. Cell sheetswere rinsed from the CellCrown insert with additional medium and imagedon an Olympus IX83 fluorescent microscope.

Statistical Methods

All data are presented as mean±standard deviation. Statisticalsignificance was calculated by performing one-way ANOVA analysisfollowed by Tukey's multiple comparison in GraphPad, Prism Software.Differences are considered significant for p<0.05.

The results of the experiments are now described.

Characterization of PNIPAAm/PCL Fibers

To generate PNIPAAm/PCL co-fibers, a solvent combination—methanol andchloroform—that dissolves both PNIPAAm and PCL was identified byreferring to previous reports on PCL solubility in electrospinningsolvents (Bordes et al., 2010, Int. J. Pharm., 383:236-243). SEM imagingconfirmed fiber formation (FIG. 1A). Eletrospinning PCL-only andPNIPAAm-only fibers in methanol and chloroform was attempted; however,fiber formation and alignment was poor compared to the PNIPAAm/PCLblended fibers. Consequently, PCL-only and PNIPAAm-only fibers wereelectrospun using HFP and methanol, respectively, following previousreports (Lee et al., 2016, Adv. Healthcare Mater., 5:781-785; Nam etal., 2008, J. Appl. Polym. Sci., 107:1547-1554). Interestingly, only the100% PNIPAAm fibers exhibited a flat, ribbon-like morphology, as waspreviously reported for PNIPAAm fibers electrospun by Rockwood et al(Rockwood et al., 2008, Polymer, 49:4025-4032). Average fiber diametersranged from 1 to 3 μm, with PCL-only fibers being the smallest diameterand PNIPAAm-only fibers having the largest diameter (FIG. 1B).Orientation analysis confirmed fiber alignment, with all conditionshaving an orientation index greater than 0.65, with 0% and 25% PNIPAAmfibers having significantly greater orientation indices than fiberscontaining 50% or more PNIPAAm (FIG. 1C). Differences in diameter andfiber orientation between PCL-only, PNIPAAm-only, and the PNIPAAm/PCLfibers may be attributable to the solvent of choice, which affectssolution viscosity—a parameter known to largely determine fiber diameter(Thompson et al., 2007, Polymer, 48:6913-6922). Furthermore, thesolution viscosity is also influenced by the relative amounts of PNIPAAmand PCL, as their respective molecular weights, effective chain lengths,and solubility in methanol and chloroform differ. It was observed thathigher PCL-content fibers could be electrospun from solutions for whichthe combined polymer concentration of the solution was lower (Table 1).For example, 90% PNIPAAm fibers were electrospun from a solution of 18%(wt/v) of 9:1 PNIPAAm:PCL whereas 25% PNIPAAm fibers were electrospunfrom a solution of 12% (wt/v) of 1:3 PNIPAAm:PCL.

FTIR spectroscopy confirmed that relative polymer compositions ofPNIPAAm/PCL fibers followed the starting PNIPAAm concentration (FIG.2C), as PCL-specific peaks increased with PCL-content andPNIPAAm-specific peaks increased with PNIPAAm content. The PCL-only (0%PNIPAAm) fibers show a strong peak at 1727 cm⁻¹ indicating carbonylstretching, which is reduced as PNIPAAm content increases and is absentfor the 100% PNIPAAm fibers. Similarly, PNIPAAm-only fibers (100%PNIPAAm) show strong peaks at 1626 and 1559 cm⁻¹ for amide groupvibrations that become less strong as PNIPAAm content decreases and arecompletely absent for the 0% PNIPAAm fibers. Additional absorbance peaksfor each polymer are listed in FIG. 7 (Beattie et al., 2014, Phys. Chem.Chem. Phys., 16:25143-25151; Elzein et al., 2004, J. Colloid InterfaceSci., 273:381-387; Dybal et al., 2009, Vib. Spectrosc., 51:44-51).

To further confirm relative PNIPAAm content and evaluate the potentialof PNIPAAm/PCL fibers for cell sheet detachment via PNIPAAm dissolution,PNIPAAm/PCL fibers were immersed in room temperature (approximately 20°C.) water to dissolve out PNIPAAm (FIG. 3A through FIG. 3C). Fibers witha considerable amount of PCL showed little mass loss (less than 5% theoriginal weight) whereas high-content PNIPAAm fibers lost more than 50%their original mass (FIG. 3A). Observations of PNIPAAm/PCL fiber areaand axial length changes before and after wetting supported the massloss data: high PNIPAAm-content fiber area contracted more than 55%whereas high PCL-content fibers did not contract but instead slightlyswelled (FIG. 3B). Furthermore, 75% and 90% PNIPAAm fiber contractionwas uniaxial, perpendicular to fiber orientation (FIG. 3C and FIG. 11).As expected, 100% PNIPAAm fibers completely dissolved, preventingmeasurements of mass loss and changes in area and axes length. For highPCL-content fibers, the percent mass lost does not match the startingpercent PNIPAAm content, indicating that the PCL protects PNIPAAm fromdissolving.

Relative hydrophobicity of the PNIPAAm/PCL fibers was determined bymeasuring advancing water contact angle above 32° C., showing that dryand wetted 100% PNIPAAm fibers (θ_(adv)=88.0° and 55.3°, respectively)were significantly less hydrophobic than 0% PNIPAAm fibers(θ_(adv)=120.7° and 107.1°, FIG. 4A, FIG. 4B, and FIG. 12). In fact, alldry, high PCL-content fibers (0%, 25%, 50% PNIPAAm) were allsignificantly more hydrophobic than 100% PNIPAAm fibers withθ_(adv)>120°. Because PNIPAAm undergoes a coil-to-globule transition atits LCST, it does not become truly hydrophobic above its LCST; rather,the hydrophobic domains are exposed to the aqueous solution, enablingprotein adsorption (Pelton, 2010, J. Colloid Interface Sci.,348:673-674). Evaluations of relatively thick and thin layers ofPNIPAAm-grafted surfaces found that Oath, decreased with thickness,meaning thicker PNIPAAm surfaces were less hydrophobic (Yamato et al.,2007, Prog. Polym. Sci., 32:1123-1133). Because biomaterialhydrophobicity is an indicator of the degree of protein adsorption, lowhydrophobicity may impair cell attachment and spreading.

The mass loss, area contraction, and advancing water contact angle dataare largely consistent in that the high PCL-content fibers behavesimilarly and that significant differences are observed for highPNIPAAm-content fibers. Significant mass loss and area contraction fromPNIPAAm dissolution starts to occur with 75% PNIPAAm. A possibleexplanation for these data is that the PNIPAAm/PCL fibers may have acore-sheath architecture, with a PNIPAAm core and PCL sheath. This hasbeen previously observed for PNIPAAm and PCL blended fibers electrospunin dimethylformamide (DMF) and chloroform, although with a PNIPAAmsheath around a PCL core (Chen et al., 2010, Chem. Mater.,22:4214-4221). Chen et al. proposed a thermodynamic argument: becauseDMF was a better solvent for PNIPAAm than for PCL and because DMF had amuch lower boiling point than chloroform, the DMF evaporated first,leaving PNIPAAm on the exterior. In the present example, methanol'sboiling point (64.7° C.) is slightly higher than chloroform's boilingpoint (61.2° C.), and methanol is a good solvent for PNIPAAm but a badsolvent for PCL. Applying the same thermodynamic argument, the PCLshould be dissolved in the chloroform portion, which would evaporatefirst to leaving PCL on the exterior of the fibers. For high PCL-contentfibers, the PCL may form an entire sheath around the PNIPAAm; however,as PNIPAAm content increases, there would not be enough PCL to protectPNIPAAm from dissolution, as observed by the mass loss and contact angledata.

Cell Viability and Alignment on PNIPAAm/PCL Fibers

After confirming the relative PNIPAAm content, the behavior of NIH 3T3fibroblasts on PNIPAAm/PCL fibers was assessed. Given PNIPAAm's relativelow hydrophobicity as indicated by advancing water contact angle,PNIPAAm/PCL fibers were coated with a 1:50 GFR-Matrigel dilution andpre-treated fibers with FBS prior to seeding, as recommended byHaraguchi et al. (Haraguchi et al., 2012, Nat. Protoc., 7:850-858);other groups have combined PNIPAAm with gelatin (Zhao et al., 2016, J.Nanosci. Nanotechnol., 16:5520-5527), chitosan (Wang et al., 2009, J.Mater. Sci.: Mater. Med., 20:583-590), fibronectin (Takahashi et al.,2011, Biomaterials, 32:8830-8838; Akiyama et al., 2004, Langmuir,20:5506-5511), collagen (Moran et al., 2007, J. Biomed. Mater. Res.,81A:870-876), poly-L-lysine, and laminin (Moran et al., 2007, J. R.Soc., Interface, 4:1151-1157) to improve cell adhesion.

MTS assay 24-hours post-seeding demonstrated that cells attached andwere viable on PNIPAAm/PCL fibers as compared to the PCL-only (0%PNIPAAm) control (FIG. 5A through FIG. 5C). However, cells weresignificantly less viable (60% relative to PCL-only control) on 100%PNIPAAm fibers. To visualize cytoskeletal actin, cells were stained withrhodamine phalloidin 24-hours post-seeding on PNIPAAm/PCL fibers. Fiberscontaining PCL (0%, 25%, 50%, 75%, and 90% PNIPAAm) showed robustspreading and significant cell alignment in a preferred direction (FIG.5B and FIG. 5C). On 100% PNIPAAm fibers, cells showed notably lessspreading and grew in clusters, which is in line with the relativelypoor cell viability observed on these fibers. As PNIPAAm is clearly nottoxic to cells, as indicted by comparable cell viability on 0%, 25%,50%, 75%, and 90% PNIPAAm fibers, it is likely that cell attachment wasaffected by poor adhesion protein adsorption due to its decreasedhydrophobicity. This is consistent with the advancing water contactresults and previous reports that protein adsorption onto PNIPAAm,especially thick PNIPAAm films (>15-20 nm), is severely limited (Akiyamaet al., 2004, Langmuir, 20:5506-5511; Moran et al., 2007, J. R. Soc.,Interface, 4:1151-1157). Understandably, coating with cell adhesionproteins (Haraguchi et al., 2012, Nat. Protoc., 7:850-858) and graftinggelatin to PNIPAAm (Zhao et al., 2016, J. Nanosci. Nanotechnol.,16:5520-5527) has been used to improve cell attachment to and spreadingon PNIPAAm surfaces. Representative histograms of cell angle show thatcells had relatively high alignment on high-content PCL fibers (FIG. 9).Although less aligned, cells on 75% and 90% PNIPAAm fibers had apreferred angle to which the cells aligned. The actin ridges thatappeared on 75% and 90% PNIPAAm fibers is likely due to PNIPAAmcontracture as the fibers had to be removed from the CellCrown insertsfor imaging. This phenomenon affected the orientation analysis as theseridges, which are perpendicular to cell orientation angle, dampened thepreferred cell angle peak.

Cell Sheet Detachment

Detachment of aligned fibroblast cell sheets was attempted by incubatingcell-seeded PNIPAAm/PCL fibers in cold medium. Following previousreports on cell sheeting (Takahashi et al., 2011, Biomaterials,32:8830-8838; Haraguchi et al., 2012, Nat. Protoc., 7:850-858) cellswere seeded at an ultra-high density—630,000 cells per cm²—to ensuresufficient cell-cell adhesion and ECM deposition. Detachment wasinitiated by rinsing cell-seeded PNIPAAm/PCL fibers with cold medium todissolve the PNIPAAm. Cell sheet detachment from PNIPAAm/PCL fibers wassuccessful for 90% PNIPAAm fibers (FIG. 6A), which occurred rapidly(less than 15 minutes). For lower PNIPAAm-content fibers, too much PCLremained preventing cell detachment. Cell sheet detachment from 100%PNIPAAm fibers was unsuccessful because the cells did not form acomplete monolayer, as indicated by the cell morphology data (FIG. 5B).

Cell sheets exhibited slight curling at the edges, evidenced by thethickening around the edges. Calcein staining confirmed that cell sheetswere viable, intact, and consisted of aligned cells (FIG. 6B and FIG.6C). Compared to previous reports of anisotropic cell sheeting fromanisotropic PNIPAAm surfaces (Takahashi et al., 2011, Biomaterials,32:8830-8838; Zhao et al., 2016, J. Nanosci. Nanotechnol., 16:5520-5527)relatively little contraction of the cell sheet was observed. This maybe due to the presence of residual PCL, which can be observed in phasecontrast images of the cell sheets (FIG. 6D and FIG. 6E).

The present data demonstrate that electrospun PNIPAAm/PCL fibers can beused to culture aligned cells, and that 90% PNIPAAm fibers can be usedfor cell detachment. This follows the original hypothesis that PNIPAAmand PCL have complementary roles and must be present in sufficientamounts. PCL encourages cell attachment, but too much PCL precludes cellsheet detachment. Likewise, PNIPAAm's low hydrophobicity limits for cellattachment but is necessary for cell sheet detachment. The cell sheetsgenerated by the present method did show some contraction. Gelatinhydrogel plungers have been previously used to prevent cell sheetcontracture and could easily be used in conjunction with the presentPNIPAAm/PCL fibers (Haraguchi et al., 2012, Nat. Protoc., 7:850-858).3T3 fibroblasts were used as a proof of principle to demonstrate thatPNIPAAm/PCL fibers can generate cell sheets. The present system can beused with other cell types to possibly generate more complex tissuestructures, such as blood vessels with better cellular architecture ofthe tunica media. Thus, electrospun PNIPAAm/PCL fibers, which are simpleand relatively inexpensive to produce, have the potential to be used togenerate anisotropic cell sheets that can either enable analyses thatare typically precluded by the use of plates or biomaterial scaffolds orbe used to create tissue-like constructs for in vivo transplantation(Shimizu et al., 2002, Circ. Res., 90:e40; Yang et al., 2007,Biomaterials, 28:5033-5043).

In summary, a simple, inexpensive, and low-resource system to generatecell sheets has been developed. PNIPAAm and PCL were successfullyelectrospun to generate aligned PNIPAAm/PCL blended fibers on which 3T3fibroblasts were cultured. Cell viability and cell alignment wasobserved on PCL and PNIPAAm/PCL fibers whereas cell viability and cellalignment was impaired on 100% PNIPAAm fibers.

Detachment of viable cell sheets by incubation with room temperaturemedium was successful for 90% PNIPAAm fibers; cell sheets did not detachfrom fibers containing less PNIPAAm and cells did not form a contiguousmonolayer on 100% PNIPAAm.

Example 2 Cardiac Differentiation on PNIPAAm/PCL Fibers for Cardiac CellSheeting

Mouse embryonic stem cells (mESCs) were differentiated to cardiomyocytes(CMs) on select PNIPAAm/PCL fibers, were characterized for contractileand electrophysiological function, and were compared with CMsdifferentiated on unaligned PNIPAAm/PC fibers and 2D mESC-derived CMmonolayers as controls. MHC+mESCs were used to facilitate CMvisualization.

Cardiac Differentiation

A comprehensive analysis is conducted for expression ofSarcomeric/cytoskeletal genes (MYH6, MYH7, MYL2, MYL7, TNNT2, TNNI3,ACTN2), calcium-handling genes (CASQ2, RYR2, SLC8A1, ATP2A3, ATP2A2,PLN), ion channels (CACNA1C, HCN4, KCNJ2, KCNJ3), and transcriptionfactors (GATA4, NKX2.5) by qRT-PCR at early and late time points (days12 and 24, respectively) to evaluate relative maturity (n=3/group with 2technical replicates).

Cell Alignment

Cells on PNIPAAm/PCL fibers and on detached sheets are stained withanti-cardiac troponin T (cTnT) to visualize CM cytoskeletons, and imagesare analyzed in ImageJ to determine CM orientation before and afterdetachment (4 ROIs/sample, n=3). Differences in cell alignment have beenobserved on aligned and unaligned fibers as cells differentiate. Fibersare stained with fluorescein-conjugated gelatin prior to cell seeding toconfirm that cell orientation follows fiber orientation (4 ROIs/sample,n=3).

Contractility

Brightfield or fluorescent time series images of CMs (aligned andnon-aligned cardiac cell sheets at 24 hours post-detachment andage-matched monolayers) are analyzed using PIVlab (an open source MATLABapplication analyzing particle image velocimetry) to determinecontraction/relaxation velocities, contraction direction, and magnitude,similar to other published work (Lee S et al., Biomaterials, 2017,131:11-20) (4 ROIs/sample, n=3/group). Using such analysis on time lapseimages of mCherry-expressing CM differentiated on aligned PCL fibers,contraction directionality was observed (FIG. 16A, FIG. 16B). Thisapproach allows for the measurement of contraction on the single celllevel. For tissue level contraction, a custom build cantilever bendingtest is used (FIG. 17). Cell sheets are attached to a testing clamp andsubmerged in Tyrode's solution. Cardiomyocyte contraction changes theposition of the cantilever, and this change in length is recorded.Changes in length can then be used to determine contraction forces(n=6). Preliminary data comparing changes in length of aligned andunaligned PCL seeded fibers showed that CMs contraction on alignedfibers caused a 10% displacement while unaligned seeded fibers had nodetectable displacement above noise levels (evaluated by unseeded fibercontrols).

Action Potential Propagation

To assess CM action potentials, intracellular calcium flux is monitoredusing the calcium-sensitive fluorophore Fluo-4, AM. Time-lapse images(up to 30 frames/second) are captured on an Axio Observer Z1 SpinningDisc confocal microscope; 3 regions of interest (ROIs) are analyzed todetermine synchronicity for cells on non-aligned and aligned fibers. Thedata demonstrates that cell alignment promotes synchronized beating(FIG. 14). Further analysis in MATLAB determines percent beating area aswell as action potential propagation velocity and direction forspontaneous beating by spatial and temporal mapping of calcium peaks. Toassess intracellular calcium dynamics, cells are paced from 0.5-5 Hz toevaluate changes in the action potential duration (specifically APD50,interval from time from 50% peak to time 50% baseline, with 4ROIs/sample, n=3/group).

Drug Responsiveness

Cell sheets and monolayers are treated with 1 μM isoproterenol,β-adrenoreceptor agonist, and 1 μM verapamil, a calcium channel blocker,to observe changes in beating rate (4 ROIs/sample, n=3/group).

Action Potential Profiles

Cardiac tissue sheets are attached to microelectrode arrays to measureaction potential profiles across the entire cell sheet. Action potentialamplitude, upstroke velocity, and resting membrane potential aredetermined (in response to chronotropic drugs, described above; n=6).

The results are now described.

Successful intact detachment of cell sheets yield viable (more than 90%live cells), beating cell sheets (at least 80% MHC+) and demonstrateimproved contractile and electrophysiological function over isotropiccell sheets and 2D monolayers. Following the preliminary data, cardiaccell sheets from aligned PNIPAAm/PCL fibers have better synchronicity(i.e., less beating variation) than sheets from unaligned fibers.Alignment improves CM function by increasing contraction/relaxationvelocity, action potential propagation velocity and persistence, anddrug responsiveness. For cell sheet formation, PNIPAAm/PCL fibersgenerally should consist of primarily PNIPAAm but have a sufficientamount PCL to allow and sustain cell attachment. Consequently,PNIPAAm/PCL fibers with high PCL content may not generate cell sheets ofcomparable performance.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

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 8. A method of making an anisotropic cellsheet, comprising the steps of: electrospinning a solution comprisingpoly(N-isopropylacrylamide) (PNIPAAm) and poly(caprolactone) (PCL) togenerate a fiber mat having fibers in substantially parallel alignment;culturing cells on the fiber mat in an environment above about 32° C. toform an anisotropic sheet of cells attached to the fiber mat; andintroducing the fiber mat to an aqueous environment below about 32° C.to release an intact anisotropic sheet of cells.
 9. The method of claim8, wherein the solution comprises a PNIPAAm and PCL mixture having aPNIPAAm to PCL ratio of between 50% (1:1 PNIPAAm:PCL) to 99% (99:1PNIPAAm:PCL).
 10. The method of claim 8, wherein the PNIPAAm to PCLratio is 90% (9:1 PNIPAAm:PCL).
 11. The method of claim 8, wherein thePNIPAAm to PCL ratio is 75% (3:1 PNIPAAm:PCL).
 12. The method of claim8, wherein the solution comprises 10% to 20% wt/v of the PNIPAAm and PCLmixture dissolved in a mixture of methanol and chloroform.
 13. Themethod of claim 8, wherein the electrospinning is performed using arotating mandrel.
 14. The method of claim 8, wherein the fibers have aPNIPAAm core and a PCL shell.
 15. The method of claim 8, wherein thefibers comprise PNIPAAm fibers and PCL fibers.
 16. The method of claim8, wherein the fibers have a diameter between about 1 and 3 μm.
 17. Themethod of claim 8, wherein the fiber mat is wetted with an aqueoussolution above about 32° C. prior to the step of culturing cells. 18.The method of claim 8, wherein the fiber mat is pretreated with a cellattachment enhancing composition prior to the step of culturing cells.19. The method of claim 18, wherein the attachment enhancing compositioncomprises at least one component selected from the group consisting of:Matrigel, fetal bovine serum, gelatin, chitosan, fibronectin, collagen,poly-1-lysine, and laminin.
 20. The method of claim 8, wherein the cellsare selected from the group consisting of: urothelial cells, mesenchymalcells, muscle cells, myocytes, fibroblasts, chondrocytes, adipocytes,fibromyoblasts, ectodermal cells, hepotocytes, Islet cells, parenchymalcells, osteoblasts, nerve cells, and stem cells.
 21. The method of claim8, wherein the fiber mat is introduced to an aqueous environment belowabout 32° C. by immersion or by rinsing.
 22. An anisotropic cell sheetformed by the method of claim 8.